A process of suicidal cell death, termed apoptosis, is vital for normal physiology because it regulates cellular turnover and preserves tissue vitality. Apoptosis is excessive in neurodegenerative diseases, immunodeficiency, etc, and is disrupted in diseases such as autoimmunity and cancer. Chemotherapeutic agents such as glucocorticoids (GCs), used in T cell leukemias, arrest cells in G1 phase of the cell cycle and induce apoptosis by altering expression of key growth regulatory/death inducing genes. The long-term goal of this PI is to understand the molecular pathway for apoptosis, emphasizing on gene regulatory changes. Using a pair of human T lymphocytic leukemia sister cell lines, CEM-C-14 and CEM-C1-15, which are sensitive and resistant, respectively to GC-evoked apoptosis, the PI has obtained preliminary data correlating the up regulation of transcript and protein levels of the anti proliferative gene, Btg1, to G1 arrest and apoptosis. Activation of the cAMP pathway sensitizes the resistant cells to GC-evoked responses, including BTG1 up regulation, G1 arrest and apoptosis. While several other gene products are also implicated in GC-evoked apoptosis of leukemic T cells, in this proposal we will focus on evaluating the relative contribution of BTG1 in modulating the process, its mode of action, and its relationship to other changes triggered by GCs. Specific Aim 1a will evaluate the ability of GCs to induce transcriptional activity from the Btg1 promoter using a promoter- luciferase reporter construct transfected in CEM-C7-14 and CEM-C1-15 cells, and will use actinomycin D and cycloheximide to determine whether GCs differentially affect the stability of Btg1 mRNA and BTG1 protein, respectively, in the two cell lines. Specific Aim 1b and c will assess the ability of GCs to alter proteasome-mediated degradation of BTG1, or alter its nuclear localization in the two cell lines. In Specific Aim 2 BTG1 expression will be repressed in CEM-C7-14 cells by siRNA to evaluate the dependence of G1 arrest and/or apoptosis on BTG1 expression, and to understand the relationship between BTG1 and expression of various growth inhibitory and apoptotic modulators such as cyclin D3, p27kip1, c-Myc, c-Jun, and Bcl-2 family members. BTG1 has been reported to bind to nuclear receptors, similar to the GC receptor (GR), and facilitate their ability to regulate gene transcription. In Specific Aim 3, the ability of BTG1 to bind to and modulate the activity of GR will be evaluated using coimmunoprecipitation assays, and transient cotransfections of GR, BTG1 and a GR-driven luciferase reporter construct, pHHLuc in Jurkat cells. These studies will significantly advance our understanding of the mechanisms of T-cell apoptosis, and the function of BTG1. 7. NARRATIVE: Apoptosis is a form of physiological cell death essential for maintaining normal health, and is triggered by altered expression or function of multiple genes, one of them being Btg1, a gene that has been shown to inhibit cell proliferation. Diseases such as Alzheimer's, neurodegeneration, autoimmunity, immunodeficiency, osteoporosis, cancer, etc are conditions where apoptosis may be a contributing factor. This application proposes studies to understand the role of Btg1 in growth inhibition and apoptosis of T-leukemic cells. [unreadable] [unreadable] [unreadable]